Method of filling a well in a substrate

ABSTRACT

The invention relates to a method of filling a well (including a channel) in a substrate. In accordance with the invention liquid is applied on a substrate comprising a well on a position that does not coincide with the well, and the well after filling is sealed with a cover means, wherein liquid is applied on a position between the front of the cover means and the well that is not covered by the cover means, and with the aid of the cover means the liquid is pushed into the well.

The present invention relates to a method of filling a well with aliquid, wherein a liquid is applied on a substrate comprising a well ona position that does not coincide with the well, and the well afterfilling is sealed with a cover means.

Such a method is generally known for filling micro arrays that are usedfor various assays, such as enzyme and DNA assays. Because of the smallamount of liquid in a well the effect of evaporation is relativelylarge. In order to prevent evaporation, a cover means such as a coverslip may be placed over the wells.

A problem is that before taking a measurement several operations have tobe carried out, e.g. filling a well with a metered amount of liquid,covering the well filled with liquid (all this preferably withoutdeveloping air bubbles), and starting the measurement. This latter oftenrequires time consuming operations, since for various measuringtechniques such as optical measuring techniques there is only littlespace around a substrate and for this reason it is often impossible tofill the substrate while mounted. In addition, for many assays thereaction will commence as soon as the well is filled with liquid.

It is the object of the invention to provide a method of the kindmentioned in the preamble by which it is possible to commence measuringmore quickly.

To this end the method according to the invention is characterized inthat

-   -   to fill the well, liquid is applied to a first place located        between a second place where a front side of the cover means        applied on the substrate is situated and the well not covered by        the cover means,    -   the front side of the cover means is moved in the direction of        the liquid that was applied and of the well, thereby taking with        it the liquid with which the well is being filled, and    -   after the front side of the cover means has passed the well, the        cover means is brought into a third stationary position in order        to seal the liquid-filled well.

Surprisingly it is possible to push a liquid to be introduced into awell forward, to fill the wells quickly and to seal them. All this isdone with one single operation, which is also favourable for optional(semi-)automated application of the method according to the invention.The present invention makes it possible to carry out a quantitativeassay on a sample, without having to accurately pipette the volume ofthe sample. After filling and covering a well in accordance with theinvention, the well contains a precisely known amount of liquiddetermined by the well's dimensions. By departing from a relativelylarge volume with relatively small surface, evaporation is reduced.Immediately after the moment a well is filled, it is already sealed bythe cover means, so that this also reduces the effect of evaporation.This is clearly different to filling individual wells with sample liquidby means of a pipette, electrospraying and the like. In the presentapplication a well is understood to be any recess in the substrateirrespective of its shape. This definition also includes elongatedwells, which may also be referred to as channel. In essence, suchchannels run parallel with the surface of the substrate.

Preferably a cover means is used having a thickness of at least 0.5 mm,more preferably at least 0.8 mm, and most preferably at least 1.2 mm.

Such a cover means ensures that the liquid is pushed forwardeffectively. At the same time, the rigidity of the cover means increaseswith the increasing thickness so that the cover means adheres better tothe substrate.

If the liquid is hydrophilic, a cover means is used in accordance with apreferred embodiment, whose side facing away from the substrate near thefront of the cover means is hydrophobic and vice versa.

This will help to prevent that part of the liquid moves over the top ofthe cover means instead of being pushed forward. This embodiment isespecially effective if a thin cover means is being used.

According to a preferred embodiment, a cover means is then used whosehydrophilic side facing the substrate possesses at one of its ends asurface with a hydrophobic portion.

The effect of such a surface portion at the end that before sliding isthe closest to the well, is that less (aqueous) liquid is necessary tofill the well.

This surface portion may be rendered hydrophobic by, for example,silanisation but also by applying a hydrophobic paste such as siliconegrease. Such a grease does not dissolve in the aqueous liquid and hasthe further advantage that it can contribute to the prevention ofevaporation. In addition, it facilitates sliding a cover means over thesubstrate.

A further problem is that during and after filling the well, reagent oranalyte is carried over from one well to the other due to flow ordiffusion. Advantageously the well is filled and sealed so quickly thatthe reagents are unable to escape from the well. Advantageously at leastin a third position the cover means is pressed to the substrate with aforce of at least 1 kg/cm².

This reduces transfer of liquid or reagents by flow and/or diffusion. Itwas also shown to be possible to apply such a force, and even a force of15 kg/cm² during movement from the first position to the third position.This further helps to limit the transfer of liquid or reagents by flowand/or diffusion.

Preferably the cover means used is a cover means of which the edge ofthe side facing the substrate and the front side of the cover means isrounded off over at least a portion thereof, and the liquid in contactwith this rounding off is moved in the direction of the applied liquidand the well, carrying along the liquid with which the well is to befilled.

Such a rounding helps to carry the liquid along and facilitates fillingthe well. Such a rounding is particularly suitable for cover meanswherein the liquid is in contact with the full height (thickness) of thecover means. The edge formed between the side facing away from thesubstrate and the front side is in such a case preferably substantiallystraight, that is to say it forms a sharp angle, which due to theeffects of cohesion is not readily passed by a liquid.

Advantageously, on a single substrate a first well is filled with afirst liquid and a second well with a second liquid, the first liquidand the second liquid are applied on respective non-overlapping firstplaces, and the cover means is provided with an indentation between thenon-overlapping first places.

Instead of this or in addition thereto a first well on a singlesubstrate is filled with a first liquid and a second well with a secondliquid, the first liquid and the second liquid being applied onrespective non-overlapping first places, and at least one organ chosenfrom i) the upper side of the substrate and ii) the lower side of thecover means between the first non-overlapping places is provided with araised barrier while the other organ in the same position has acomplementary groove in which the barrier can be received, so that whenfilling the first and second wells, the barrier is slid through thegroove.

Both methods allow wells of a substrate to be filled with differentliquids.

According to a favourable embodiment, the cover means comprises at leasttwo front sides, at least one front side being formed by a wall of agroove provided at right angles to the direction of movement at theunderside of the substrate, and a first well on a single substrate beingfilled with a first liquid and a second well with a second liquid, thefirst liquid and the second liquid being applied on respectivenon-overlapping first places, such that the well of the first liquid andthe well of the second liquid are on the second first place, after whichthe cover means is placed on the substrate and subsequently moved overthe substrate in order to fill the first well and the second well.

In this way wells that are substantially in each other's extendeddirection, can be filled simultaneously with different liquids.

In order to effectively seal a well after the cover means has been movedto the third position, it is preferred for both the substrate and thecover means to be provided with functional groups capable of forming aphysical or chemical bond with the functional groups with which they arein contact.

For example, one component may be provided with amino groups and theother with epoxy groups. Various functional groups can be found quiteeasily in the literature relating to immobilisation of proteins andother molecules on a surface, therefore the ordinary person skilled inthe art requires no further explanation.

The invention also relates to a cover means for the application of themethod according to one of the claims 11 to 14, as well as a substratefor the application of the method according to one of the claims 11 to14.

The present invention will now be elucidated with reference to anexplanatory embodiment and the drawing, in which

FIGS. 1 a and 1 b respectively show a side view of a substrate withcover means according to the invention, and a top view of the covermeans and substrate;

FIGS. 2 a and b show a cross-sectional side view of a substrate withcover means according to the invention for the application of differentliquids in different wells (before and after); and

FIG. 3 shows a top view of a substrate with cover means according to theinvention.

FIG. 1 shows a substrate 1 provided with two arrays 2, 3, whicharrays-2, 3 consist of wells 4. The substrate 1 is also provided withtwo grooves 5′, 5″, which like the wells 4 have conveniently beenprovided in the substrate 1 by means of etching. In accordance with onepossible embodiment of the invention, the grooves 5′, 5″ are suitablefor introducing liquid A into the wells 4. Instead of that, or inaddition thereto, it is possible in accordance with an alternativeembodiment to provide a linear coating, e.g. a hydrophilic coatinghaving a greater affinity for the liquid. Instead of that, this placemay be delimited by two lines of coating for which the liquid has lessaffinity. In the case of an aqueous liquid being applied it is possible,for example, to use Teflon spray. This may be done quite easily byprotecting the place where the liquid is to be applied by means of, forexample, a rectangular piece of paper. After the liquid A has beenapplied, a cover means 6 positioned on the substrate is moved in thedirection of the wells. In FIG. 1 the wells 4 of array 2 are alreadyfilled.

The side of the cover means 6 facing the substrate and the side of thesubstrate 1 facing the cover means 6 are completely smooth so that oncelaid on the substrate, the cover means 6 is equidistantly moveable inrelation to the substrate 1, and the sample liquid A can be pushed fromthe grooves 5′, 5″ into the wells 4. After that the filled wells 4 areimmediately sealed by the cover means 6. The surplus liquid A can beblown away with the aid of an air gun (pressure approximately 4-5 bars).Prior to that and preferably during the removal of surplus liquid, aclamping force may be exerted if necessary, which is suitably 2-3kg/cm². The removal of the liquid ensures that any surface tensionalforces do not cause this liquid to be drawn between the cover means 6and substrate 1, which would increase the thickness of the liquid layerbetween the cover means 6 and the substrate 1. A very thin liquid layerguarantees that even without a clamping force the cover means will sealthe wells 4 by means of adhesion for a measuring time of, for example,5-20 minutes. For the performance of measurements, the wells 4 maycontain reagents that may dissolve in the liquid A with which the wellshave been filled. The presence of a thin layer of liquid minimises theexchange (by diffusion) of any possibly present reagent from one well toan adjacent well. Finally, evaporation of a liquid during measurement iseffectively avoided with the method described.

The cover means 6 and the substrate 1 are also clean (free of dust) and,in the case of aqueous liquids A, preferably at least on the sides ofthe substrate 1 and cover means 6 facing each other, hydrophilic.Nevertheless, in the case of aqueous liquids A the end of the side ofthe cover means 6 facing the substrate 1 is in accordance with apreferred embodiment close to the wells 4 hydrophobic. In this way itcan be prevented that relatively much liquid sample A is required forfilling the wells 4. According to a simple embodiment, the end of theside facing the substrate 1 is made hydrophobic with the aid of siliconegrease. The amount of silicone grease used is kept to a minimum (i.e. avery thin coating is applied). The use of a groove 5 is not aprerequisite and may even be undesirable. The liquid A is preferablyapplied to the frontal side of the cover means 6, oriented toward to thewells 4. In this way it is still possible to spread the liquid Atransversely. Said frontal side is preferably hydrophilic.

The rate at which the cover means 6 is to be moved in relation to thesubstrate 1 depends on the materials used and on the liquid A used aswell as the components, such as surfactants, comprised therein.Nonetheless, a person skilled in the art can easily determine a suitablerate. If the percentage of wells 4 comprising entrapped air is higherthan desirable, the rate must be reduced or wells with a rounded or lesssteep wall must be used (these fill more easily). The substrate 1 andthe cover means 6 may be manufactured from any material that isessentially inert (in relation to the liquid A), such as polystyrene,glass or materials that can be treated using techniques known from thesemiconductor industry.

Near the substrate side at the front end, the cover means 6 possesses anedge 7, which is preferably provided with a rounding (indicated by thedotted line). This allows the wells 4 to be effectively filled withliquid. A practical method of filling wells is performed as follows:

-   -   1) A Pyrex glass (1×0.8 cm) having a thickness of 1 mm is        cleaned by treating it with a detergent and rinsing well with        demineralised water.

2) The Pyrex glass is placed on a semiconductor substrate. Thesemiconductor substrate is provided with a hydrophilic SiO₂- andpreferably a silicon nitride layer. The semiconductor substrate isprovided with wells of 400×400 μm having a depth of 50 μm.

-   -   3) By means of capillary action 0.1 to 0.2 μl water is        introduced between the Pyrex cover glass and the substrate. The        presence of such a minimal amount of liquid causes the Pyrex        cover glass to adhere to the substrate by means of suction. It        is difficult to remove but can slide.    -   4) A sample liquid (1 μl) is pipetted in front of a row of wells        at a distance from the Pyrex cover glass.

5) The Pyrex cover glass is pushed into the direction of the sampleliquid and the wells at a rate of 2-6 cm/s.

-   -   6) The cover glass is pressed onto the substrate with a force of        2-3 kg/cm² and surplus sample liquid is blown away with the aid        of an air gun (pressure 4 bars).    -   7) The substrate with cover glass are placed in a slide object        holder on an X-Y-Z-table under an objective lens.

In an experiment an array of 5×5 wells of 400×400×50 μm (total volume0.2 μl) was filled using only 0.5 μl liquid.

Experiments (colour reactions in which the wells were coated with anenzyme) showed that carry-over in a direction transversally to thedirection of movement of the cover means in relation to the substratewas minimal. There was some detectable carry-over between successivewells in the direction of movement.

The method according to the invention was shown to be suitable forfilling wells with a volatile solvent such as ethanol. This allows themethod according to the invention to be applied for combinatorialchemistry and potentially dangerous reactions that are safe to beperformed on a small scale.

To the ordinary person skilled in the art it will be obvious that it ispossible to vary the present method in many ways without departing fromthe scope of the invention. For example, in particular if a well isrelatively deep, it is advantageous to use a cover means 6 that is notstraight e.g. a serrated or fluted one, wherein the serrated or flutedrecesses take up the liquid and the cover means 6 with the recesses ispushed over the well 4. If an array 2 has to be filled, the position ofthe recesses is chosen such that they substantially coincide with thecentres of the wells. If desired, different recesses may containdifferent sample liquids so that different sample liquids can be assayedsimultaneously.

In order to apply a layer of sealing, hydrophobic paste such as siliconegrease in a defined and reproducible manner, use may be made of aspreader that is at least in one place in contact with the substratewhile being moved over the substrate in a direction of an excess ofpaste. The spreader leaves a narrow groove between the substrate and thespreader, which narrow groove (and the angle at which the spreader ismoved), determine the thickness of the layer. The spreader mayoptionally be also provided with a guide member resting against a sideof the substrate.

FIG. 2 a shows a substrate 1 provided with various wells 4. The covermeans 6 is provided with grooves 8, each of which have a front wall 9pushing the various liquids A, B, C, D forward. When the cover means 6is in the third position, the wells 4 are sealed. The wells 4 maycomprise various reagents W, X, Y, Z.

FIG. 3 shows a top view of three arrays 2, 3, 9 comprised of wells 4′,4″, 4′″, to be filled with different liquids A, B, C. In order toprevent the liquids that during the movement of the cover means arebeing spread along the front edge from coming into contact with eachother, recesses 9 are provided, so that the liquids are virtuallysubjected to different cover means 6.

The application of the liquids and the movement of the liquids ispreferably automised by means of dispensers and actuators.

1. A method of filling a well with a liquid, wherein a liquid is appliedon a substrate comprising a well on a position that does not coincidewith the well, and the well after filling is sealed with a cover means,characterised in that to fill the well, liquid is applied to a firstplace located between a second place where a front side of the covermeans applied on the substrate is situated and the well not covered bythe cover means, the front side of the cover means is moved in thedirection of the liquid that was applied and of the well, thereby takingwith it the liquid with which the well is being filled, and after thefront side of the cover means has passed the well, the cover means isbrought into a third stationary position in order to seal theliquid-filled well.
 2. A method according to claim 1, characterised inthat a cover means is used having a thickness of at least 0.5 mm,preferably at least 0.8 mm, and most preferably at least 1.2 mm.
 3. Amethod according to claim 1 or 2, characterised in that if the liquid ishydrophilic, a cover means is used, whose side facing away from thesubstrate near the front of the cover means is hydrophobic and viceversa.
 4. A method according to claim 3, characterised in that the covermeans at the hydrophilic side facing the substrate possesses at one ofits ends a surface with a hydrophobic portion.
 5. A method according toclaim 4, characterised in that the hydrophobic surface portion comprisesa hydrophobic paste.
 6. A method according to one of the precedingclaims, characterised in that at least in a third position the covermeans is pressed to the substrate with a force of at least 1 kg/cm^(2.)7. A method according to one of the preceding claims, characterised inthat the substrate comprises an array of wells, a groove is provided inthe substrate with a main directional component being at right angles tothe direction of movement of the cover means, and the liquid isintroduced into the groove prior to being transferred to the array bythe cover means moving over the groove.
 8. A method according to one ofthe preceding claims, characterised in that when the cover means is inthe third position, the liquid surrounding the cover means is removed.9. A method according to claim 8, characterised in that removal occursby means of compressed gas.
 10. A method according to one of thepreceding claims, characterised in that the cover means used is a covermeans of which the edge of the side facing the substrate and the frontside of the cover means is rounded off over at least a portion thereof,and the liquid in contact with this rounding off is moved in thedirection of the applied liquid and the well, carrying along the liquidwith which the well is to be filled.
 11. A method according to one ofthe preceding claims, characterised in that on a single substrate afirst well is filled with a first liquid and a second well with a secondliquid, the first liquid and the second liquid are applied on respectivenon-overlapping first places, and the cover means is provided with aindentation between the non-overlapping first places.
 12. A methodaccording to one of the preceding claims, characterised in that a firstwell on a single substrate is filled with a first liquid and a secondwell with a second liquid, the first liquid and the second liquid beingapplied on respective non-overlapping first places, and at least oneorgan chosen from i) the upper side of the substrate and ii) the lowerside of the cover means between the first non-overlapping places isprovided with a raised barrier while the other organ in the sameposition has a complementary groove in which the barrier can bereceived, so that when filling the first and second wells, the barrieris slid through the groove.
 13. A method according to one of thepreceding claims, characterised in that the cover means comprises atleast two front sides, at least one front side being formed by a wall ofa groove provided at right angles to the direction of movement at theunderside of the substrate, and a first well on a single substrate beingfilled with a first liquid and a second well with a second liquid, thefirst liquid and the second liquid being applied on respectivenonoverlapping first places, such that the well of the first liquid andthe well of the second liquid are on the second first place, after whichthe cover means is placed on the substrate and subsequently moved overthe substrate in order to fill the first well and the second well.
 14. Amethod according to one of the preceding claims, characterised in thatboth the substrate and the cover means are provided with functionalgroups capable of forming a physical or chemical bond with thefunctional groups with which they are in contact.
 15. A cover means tobe used with the method according to one of the claims 11 to
 14. 16. Asubstrate to be used with the method according to one of the claims 11to 14.